This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To develop a media formulation and protocol for the human ES cell culture that will improve human ES cell viability [unreadable] and promote uniformity, consistency and ease of use across a variety of users in different locations. Some of the [unreadable] specific objectives necessary to accomplish this goal are to: 1) optimize the physiochemical environment, 2) [unreadable] optimize the basal media formulation and 3) eliminate undefined media components.[unreadable] [unreadable] Human embryonic stem (ES) cells already provide a powerful research tool for understanding the human body, the [unreadable] current sub-optimal conditions for human ES cell culture place significant limitations on their use in basic research and [unreadable] on their large-scale expansion and distribution. The labor required to continuously prepare MEF feeder layers is a major [unreadable] limitation in large-scale production of human ES cells. Combined with concerns surrounding cross-species [unreadable] contamination that may arise from growth of human ES cells on murine feeder layers, the elimination of fibroblast [unreadable] feeder cells would greatly improve the efficiency and consistency of ES cell culture. We have previously reported that [unreadable] high bFGF concentrations support feeder-independent growth of human ES cells, but those conditions included poorly [unreadable] defined serum and matrix components. In the previous budget year we reported a feeder-independent human ES cell [unreadable] culture that includes protein components solely derived from recombinant sources or purified from human material [unreadable] (Nature Biotechnology, February 2006). While this culture system is efficient and effect at propagating human ES cells, [unreadable] it is cost prohibitive for most research laboratories. In this budget year, we developed and reported on (Nature Methods, [unreadable] August 2006) a modified version of our feeder-independent medium (mTeSR1) that is appropriate for use in standard [unreadable] human ES cell research laboratories. In applications where completely humanized media is not essential, the use of [unreadable] animal sourced proteins and zebrafish bFGF (zbFGF) significantly reduces the cost of preparation and enables the [unreadable] widespread adaptation of feeder-independent culture systems for human ES cell research. We also continue to optimize [unreadable] the medium to improve efficiency, conduct research to improve cloning efficiencies and identify alternate matricies to [unreadable] future improve human ES cell culture overall. This research used WNPRC stem cell resources and federally approved hES [unreadable] cell lines.